The renin-angiotensin system (RAS) is a major regulator of cardiovascular homeostasis. See, for example, Hypertension and the Angiotensin System-Therapeutic Approaches, A. E. Doyle and A. G. Bearn, ed., Raven Press, 1983, and references contained therein. In the RAS, angiotensin I (AI) is formed from angiotensinogen by the enzyme renin. AI, a decapeptide, is cleaved by converting enzyme to angiotensin II (AII), the effector molecule, which is an octapeptide. At least two compartments of AII, one localized in the plasma and the other localized in the vascular tissue, contribute towards the blood pressure elevation in various hypertensive states. An understanding of their relative contributions in these different states remains an important problem in the diagnosis and treatment of hypertension.
Experimental and clinical studies of the RAS have been greatly aided by the development of pharmacologic inhibitors which interfere at various points in the system. For example, suppression of converting enzyme activity by inhibitors such as captopril and enalapril now represents an important approach to anti-hypertensive therapy. However this enzyme is capable of hydrolyzing many peptide substrates in addition to AII, including bradykinin, substance P, enkephalins and neurotensin; V. J. Dzau, J. Cardiovascular Pharmacol., 7, S53 (1985). Therefore inhibitors of the enzyme are unlikely to be physiologically specific with respect to the RAS. A lack of physiological specificity may also apply to inhibitors of the enzyme renin, a protease whose substrate specificity has recently been recognized to comprise more than just angiotensinogen; T. Inagami, K. Ohtuski, T. Inagami, J. Biol. Chem., 258, 7476 (1983). Since AII is the primary biologically active component of the RAS, an antagonist to this hormone should represent a physiologically specific inhibitor of the RAS. Current peptide antagonists of AII, such as saralasin ([Sar.sup.1, Ala.sup.8 ] AII) are limited in their use as RA inhibitors by their inherent partial agonist properties.
No drugs which are currently used to block the RAS have the capacity to selectively neutralize plasma AII. Such a selective agent would be particularly suitable for the treatment of disorders such as renovascular hypertension and congestive heart failure. In patients with these disorders, it is desirable to neutralize the high levels of AII in the plasma without neutralizing the AII in the tissues, particularly in the kidney. This is because intrarenal AII is important in maintaining renal hemodynamics. Inhibition of the intrarenal AII, as occurs during prolonged treatment with drugs currently used to inhibit the RAS, (such as the converting enzyme inhibitor, captopril) may lead to impaired kidney function; Silas, et al., Br. Med. J., 286, 1702, (1983). A recent clinical study concluded that while converting-enzyme inhibition with drugs such as captopril and enalapril produces benefits in patients with congestive heart failure, this therapeutic approach is associated with a significant risk of hypotension, whose magnitude and duration determine whether serious end-organ (cerebral and renal) deficits will occur; Packer et al., N. Engl. J. Med., 315, 847, (1986).
Antisera directed against AII have been evaluated in the past for their effects in blocking the RAS in various normotensive and hypertensive experimental models. However, to date the data derived from immunological blockade of AII in vivo has been equivocal. Worcel et al., Suppl. Circ. Res., 26, 223, (1970), Bing and Poulsen, Acta Path. Microbiol. Scand., 78, 6 (1970), and Brunner et al., J. Clin. Invest., 51, 58 (1972) have described a blood pressure reduction in renal hypertensive rats characterized by elevated levels of plasma AII following intravenous administration of rabbit serum containing polyclonal antibodies to AII. However in most of these animals, the decreases were very short-lived. For example, Brunner et al., state that the blood pressure invariably returned to base line levels in 5 to 15 minutes. Only 1 rat in Bing and Poulsen's study reacted to an injection with a lasting (greater than 40 minutes) depression. Furthermore, studies by Hedwall, Br. J. Pharmacol., 34, 623 (1968), revealed that the blood pressure of renal hypertensive rats was uninfluenced by intravenous injection of rabbit AII antiserum. In another study, Walker et al., Proc. Fifth International Congress of Nephrology, 1115 (1972), demonstrated an inability of circulating AII antibodies in rabbits, generated by active immunization with the hormone, to reduce blood pressure.
The inconsistencies of these studies are most likely due to the presence of components in the antisera capable of affecting blood pressure independent of the RAS, and to the pressure of populations of antibodies which bind to AII but which do not prevent it from binding to its cellular receptor and triggering its physiological effects. Furthermore, these inconsistent results reflect our inability to predict whether a particular antibody or antibodies to AII will reduce the blood pressure in experimental animals by specific blockade of the RAS, and therefore, whether they have potential therapeutic utility as anti-hypertensive agents. Haber et al., Am. J. Physiol., 7, H404 (1984), summarized the state of the art by concluding the data derived from immunological blockade of AII has been equivocal.
The diagnostic utility of polyclonal antibodies to AII for measuring hormone levels from the plasma of hypertensive patients is also limited. This is because AII antisera nearly always are directed to the carboxy terminus of AII and therefore strongly cross-react with the heptapeptide and hexapeptide metabolites of AII: (des-Asp)-AII (AIII) and (des-Asp, Arg)-AII, respectively; Nussberger et al., J. Immunol. Methods, 56 85 (1983). Because these products have important activity differences compared to AII, selective measurement of AII is of considerable significance.
Kohler and Milstein, Nature, 256, 495 (1975), were the first to describe methods of making monoclonal antibodies by fusing spleen cells from an immunized mouse to a drug-resistant plasmacytoma cell line and isolating the hybrid clones by growth on selective medium. Monoclonal antibodies can overcome many of the problems associated with the use of polyclonal antisera, namely purity, specificity, homogeneity and availability.
Although the general technique of producing hybridomas is well known, there are still considerble difficulties involved in producing and selecting a hybridoma cell line which secretes antibody having a given set of desired properties.
Nussberger et al., Hybridoma, 3, 373 (1984) described the production of a monoclonal antibody to AII where the antigen used was AII coupled to thyroglobulin. However, this antibody was very similar in properties to polyclonal sera directed to AII in that it failed to significantly differentiate between AII and the smaller peptide fragments AIII and (des-Asp, Arg)-AII. It is doubtful whether this antibody with its low affinity for AII (Ka=0.3.times.10.sup.7 M.sup.-1) would lower blood pressure in experimental animals by blocking the RAS. There is no teaching or suggestion of utilizing this antibody as an AII antagonist in physiological studies.
There remains a need for a specific high affinity antagonist of AII which can neutralize its biological actions and thereby be utilized therapeutically as a specific inhibitor of the RAS. Furthermore, a specific antagonist of the RAS which can selectively inhibit plasma AII would be a highly desirable agent for treatment of renovascular hypertension and congestive heart failure. Finally there is a need for a reagent which can be utilized to measure levels of AII free from significant cross-reactivity with its metabolites.